Methods for the production of diatom biomass

ABSTRACT

The invention relates to methods of producing a diatom biomass. More particularly, the invention relates to methods to produce a diatom biomass using a continuous culture to produce a volumetric production rate of biomass of at least 20 g dry weight/L/day, wherein the culture medium is designed to provide the essential nutrients to maintain the diatom in log phase growth. In a preferred embodiment the biomass comprises highly unsaturated fatty acid.

FIELD OF INVENTION

The invention relates to methods of producing a diatom biomass. Moreparticularly, the invention relates to methods to produce a diatombiomass using a continuous culture. In a preferred embodiment thebiomass comprises highly unsaturated fatty acid.

BACKGROUND

Diatoms are a widespread group of microalgae and can be found in theoceans, freshwater, and in soils. Biomass from diatom culture (whethernaturally or commercially produced) contains a number of products ofcommercial interest including lipids, fatty acids (particularly highlyunsaturated fatty acids—HUFAs), amino acids, pigments and complexnatural products of pharmacological interest. The biomass itself alsohas dietary application (particularly in aquaculture), and the treatmentof water contaminated by phosphorus and nitrogen in aquacultureeffluent, or heavy metal (bioremediation). A further application indevelopment is the use of silicon derived from frustules (the cell wallor external layer of diatoms) in nanotechnology. However, the commercialrelevance of products from diatoms will depend on the cost of theirproduction.

Industrial fermentation is a costly process in terms of capitalequipment, nutrients, and energy, and is generally only justified when arelatively high value product is being produced in large quantities.

Different modes of culturing or fermentation are possible. The simplestmode of fermentation and the one that is almost exclusively used inindustrial processes is batch fermentation. In batch fermentation, cellsare inoculated in nutrient medium, grown for a period of time and thenharvested. Fed-batch fermentation is similar to batch but differs inthat concentrated nutrients are supplied to the culture during thegrowth period. Continuous fermentation involves the continuous harvestof culture comprising biomass and a nutrient solution from thefermentation vessel and its replacement with fresh nutrient solution.The rate of harvest in continuous fermentation is chosen so that thedensity of the cells in culture remains constant. Semi-continuousfermentation is similar to continuous fermentation except that harvestsare periodic rather than continuous. Perfusion fermentation involves thecontinuous harvest of a culture medium which contains the product ofinterest, while the cells producing the product are retained in theculture.

Where the product of interest is produced within the cells, the cellsare generally cultivated to the highest biomass densities possible toget the most efficient volumetric productivity (i.e., amount of productproduced per volume of fermentation medium per unit time), therebyminimizing the cost of production of the product of interest. However,at high biomass densities there can be difficulties in providingsufficient oxygen, nutrients and, where applicable, light. For example,in a batch or fed batch fermentation, microbial cultures are generallyin a stationary phase at the point of harvest. It is usual for themedium or feeding regime in these types of fermentations to result inthe culture being severely limited for one or more nutrients, often as ameans of inducing the formation of secondary metabolites (which areoften the products of commercial interest).

It was previously known that photosynthetic organisms (e.g., microalgae)can be grown phototrophically under continuous or semi-continuousculture conditions. Under these conditions light is used as the energysource, rather than reduced carbon. Many authors (e.g. Richardson et al.(1969) Applied Microbiology 18:245-250; Droop (1974) J. Mar. Biol. Ass.U.K. 54:825-855; Laing (1991) Lab.Leafl.MAFF Direct.Fish.Res.,Lowestoft, (67) 31 pp) have disclosed means of growing algae incontinuous or semi-continuous phototrophic culture (turbidostats). Insuch cultures, cells are generally limited by the amount of light thatis available and so culture densities are low and thus volumetricproductivity is very low. Advances in photobioreactor technology haveovercome the light limitation problem to a certain extent by narrowingthe optical plane and allowing cultures to reach higher biomassconcentrations (Zou et al. (2000) Eur. J. Phycol. 35:127-133). However,this requires an increase in surface area to volume ratio of the cultureand significantly increases the capital cost structure of the reactors.Furthermore, at these concentrations growth is slow (light limited) andthus volumetric productivity is again low. These photobioreactor systemsalso have the disadvantage that they would be uneconomic to constructfor volumes large enough to supply industrial demand and even the bestcommercially scalable photobioreactor designs still produce biomassdensities only about one-tenth that capable in a fermentor. Suchphotobioreactors are therefore generally considered to be just alaboratory tool for study of the growth of photosynthetic organisms.

Pharmaceuticals, medical foods and nutritional supplement containinghighly unsaturated fatty acids (HUFAs) are currently used to treathundreds of thousands, and potentially will soon be used to treat,millions of patients. Eicosapentaenoic acid (EPA) is a HUFA used as anactive metabolite in drug substances. Docosahexaenoic acid (DHA) alsohas great potential for use in the pharmaceutical, medical food andnutritional supplement industry. An example of their use is thetreatment or prophylactic treatment of cardiovascular disease.

HUFAs cannot be chemically synthesized de novo economically, thereforemust be extracted from a biological source. Pharmaceutical manufacturerscurrently rely on fish as the source of HUFAs for production of drugsubstances. Exclusive reliance on fish oil for such purposes, however,carries a number of serious risks to pharmaceutical manufacturers anddrug companies as well as potentially to patients receiving suchmedications. Such risks include, but are not limited to, thoseassociated with potential supply shortages, which may be financiallydevastating to drug companies as well as negatively affect patients whorely on medications for their well-being. Fish oil itself is not aspecific reference material since its composition differs dramaticallybetween different fish species. Even within a single fish species thecomposition varies from location to location and it even varies atdifferent times of the year at a single location. Such variability instarting material makes manufacturing a final pharmaceutical productvery difficult. Furthermore there is a growing concern over the toxicpollutants such as poly chlorinated biphenyls (PCBs), dioxin, and methylmercury that are showing up in fish oils. There are also widespreadconcerns over the long term sustainability of wild fish stocks andaquaculture.

There is an acute need therefore, for alternative sources of HUFAs tofish oil which are consistent, reliable and therefore amenable tocommercial and sustainable production methods. Industrial fermentationis one possible alternative for commercial production of HUFAs. However,there are a number of problems with using industrial fermentation on acommercial scale.

A number of authors have disclosed continuous culture of microorganismsfor the production of lipids (for example Hall and Ratledge (1977) App.Env. Microbiol 33:577-584; Gill et al. (1977) App. Env. Microbiol.33231-239; Ykema et al. (1988) Appl. Microbiol. Biotechnol. 29:211-218;Brown et al. (1989) J. Ferm. Bioeng. 68:344-352; Kendrick and Ratledge(1992) Appl. Microbiol. Biotechnol. 37:18-22; Papanikolaou and Aggelis(2002) Bioresouce Technology 82:43-49). Whilst some of these do disclosehigh volumetric biomass productivity, none of the organisms disclosedproduce any highly unsaturated fatty acids (HUFAs; fatty acids with 20or more carbons and 4 or more double bonds).

Other types of organisms, in particular microalgae of the genusCrypthecodinium and marine fungi in the family Thraustochytriales(including Schizochytrium species, Thraustochytrium species and Ulkeniaspecies) may be more uniquely suited to production of HUFAs, andparticularly the omega-3 DHA. Whilst these organisms produce thegreatest amounts of DHA under conditions of nitrogen limitation and arethus well suited to the fed batch cultures used in industrial processes,some authors have disclosed a continuous heterotrophic culture of thesespecies for the production of DHA. However, most of these continuouscultures were only demonstrated in the laboratory and were at lowproduction rates, which are not commercially viable.

Ganuza and Izquierdo ((2007) Appl. Microbiol. Biotechnol. 76:985-990)disclose continuous culture of Schizochytrium G13/2S for the productionof DHA. Their maximum biomass productivity occurred at a dilution of0.04 per hour and a dry weight of 7.7 g/L giving a volumetricproductivity of only 7.4 g/L/day.

Ethier et al. ((2011) Bioresource Technology 102:88-93) disclosecontinuous culture of Schizochytrium limacinum for producing DHA. Theirhighest biomass productivity was only 3.88 g/L/day.

Pleisner and Errikson(http://www.marbio.sdu.dk/uploads/MarBioShell/Pleissner%20-%20VejleNuthetalPoster.pdf accessed 8 Feb. 2013), disclosecontinuous culture of Crypthecodinium cohnii for producing DHA, but onlyachieve a biomass productivity of 12 g/L/day. Further, at thisvolumetric productivity the HUFA content of the biomass was only 1.67%,making the final extraction and processing problematic.

In contrast, Wümpelmann in WO2005/021735 discloses continuous culture ofSchizochytrium limnaceum for the production of DHA at high culturedensities and through this achieves biomass productivities in excess of100 g/L/day. The author discloses methods that are particularly suitableto producing DHA from this organism, indicating for instance thatdissolved oxygen tension should be maintained at low levels, but doesnot provide sufficient guidance about whether such conditions can evenbe used for other types of microorganisms or, if so, how such conditionsshould be varied to allow these sorts of productivities for otherorganisms with completely different environmental and nutritionalrequirements. The methods disclosed by the author are also not wellsuited for production at an industrially relevant scale. Indeed, theexamples disclosed are only carried out in 2 L fermenters in thelaboratory and the cost of using just one of the media constituents(e.g., casamino acids) as a nitrogen source would be prohibitivelyexpensive at scales of 100 L or more.

Other types of organisms, in particular the diatoms, may be more suitedfor the production of the omega-3 EPA, especially where it isadvantageous for the EPA to be produced with relatively low amounts ofDHA present.

Wen and Chen disclose the heterotrophic culture of the diatom Nitzschialaevis for the production of the omega-3 fatty acid EPA. However, intrue continuous cultures their maximal biomass productivity was only 2.8g/L/day ((2002) Biotechnol. Prog. 18:21-28), whilst with the addition ofperfusion (wherein cells and media are separated, cells are returned tothe fermenter and additional fresh media is added), this was raised to6.75 g/L/day ((2001) Appl Microbiol Biotechnol 57:316-322).

Griffiths et al. (WO2011/155852) disclosed the heterotrophic culture ofthe diatom Nitzschia laevis both in perfusion-aided continuous cultureand in true continuous culture. Whilst the authors do no not disclosethe volumetric productivities of the cultures, culture dry weights werearound 10 g/L or lower and in perfusion-aided continuous culture thetotal volume removed from the culture was one fermenter volume per day.Even if all this volume were harvest and none were perfusion, themaximum biomass productivity would be significantly higher thandescribed by Wen and Chen, but would still only be around 10 g/L/day.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a methodof producing a diatom biomass, the method comprising the step ofcontinuously culturing a diatom in a culture medium to produce avolumetric production rate of biomass of at least 20 g dry weight/L/day,wherein the culture medium is designed to provide the essentialnutrients to maintain the diatom in log phase growth.

Preferably the diatom produces at least one highly unsaturated fattyacid (HUFA).

Preferably the HUFA is selected from any one or more of EPA, DHA andARA.

Preferably the HUFA is an omega-3 fatty acid.

Preferably the diatom biomass contains a total HUFA level of at least 2%of dry cell weight of the biomass. Preferably the diatom biomasscontains a total HUFA level of at least 3% of dry cell weight of thebiomass.

Preferably the diatom biomass contains a mixture of EPA and DHA at alevel of at least 2% dry cell weight of the biomass.

Preferably the diatom biomass contains at least one HUFA at a level ofat least 2% of dry cell weight of the biomass.

Preferably the diatom biomass contains EPA at a level of at least 2% ofdry cell weight of the biomass.

Preferably the volumetric production rate of biomass is at least 25 gdry weight/L/day. Preferably the volumetric production rate of biomassis at least 30 g dry weight/L/day.

Preferably the mean volumetric production rate of biomass is at least 20g dry weight/L/day over a period of at least 72 hours. Preferably themean volumetric production rate of biomass is at least 20 g dryweight/L/day over a period of at least a week. Preferably the meanvolumetric production rate of biomass is at least 20 g dry weight/L/dayover a period of at least a month. Preferably the mean volumetricproduction rate of biomass is at least 20 g dry weight/L/day over aperiod of at least 2 months.

Preferably the method does not include a perfusion culture of thecultured diatoms.

Preferably the diatom is a Nitzschia species. Preferably the diatom isNitzschia laevis. Preferably the diatom is Nitzschia laevis strainIn1CS20.

Preferably the diatom is a Cyclotella species.

Preferably the diatom is a Phaeodactylum species.

Preferably, the diatom is cultivated heterotrophically.

Preferably the culture medium comprises a reduced carbon source.

Preferably the reduced carbon source is selected from any one or moreof: glucose, fructose, high fructose corn syrup, monosaccharides,disaccharides, alcohols, acetic acid or its salts.

Preferably a source of silicate is added continuously during the courseof the culture.

Preferably the source of silicate is an alkali metal silicate.

Preferably the alkali metal silicate is sodium silicate or potassiumsilicate.

Preferably the sodium silicate is added continuously at a level ofbetween 20 and 120 mg sodium metasilicate pentahydrate for every gramdry cell weight of biomass produced in the culture.

Preferably the silicate is added at between 40 and 100 mg sodiummetasilicate pentahydrate per gram of dry cell weight of biomassproduced. Preferably the silicate is added at 75 mg sodium metasilicatepentahydrate per gram of dry cell weight of biomass produced.

Preferably the culture pH is maintained within the range 7.0 to 9.0.Preferably the culture pH is maintained within the range 7.5 to 8.5.Preferably the culture pH is maintained within the range 8.0 to 8.5.

Preferably the culture is maintained at a temperature of between 15 and30° C. Preferably the culture is maintained at a temperature of between20 and 25° C.

Preferably agitation and/or back pressure are provided to maintain adissolved oxygen level at or above 10% of air saturation.

Preferably the dissolved oxygen level is maintained at or above 20% ofair saturation. Preferably the dissolved oxygen level is maintained ator above 30% of air saturation. Preferably the dissolved oxygen level ismaintained at or above 40% of air saturation.

Preferably the method includes replacement of at least 50% of theculture volume with fresh medium every 24 hours.

Preferably the method includes replacement of at least 10% of theculture volume with fresh medium every 4 hours.

Preferably the method includes replacement of at least 5% of the culturevolume with fresh medium every 2 hours.

Preferably the method includes replacement of at least 0.2% of theculture volume with fresh medium every 5 minutes.

Preferably the method includes at least 500 liters of culture medium.

Preferably the method further includes separating the biomass from theculture and concentrating the biomass to produce a biomass concentrate.

Preferably the biomass is concentrated by any one or more of:centrifugation, flocculation, filtration, or floatation.

Preferably the biomass is concentrated by continuous centrifugation.

Preferably the method further includes the step of drying of the biomassconcentrate.

Preferably the biomass concentrate is dried by any one or more of: spraydrying, freeze drying, tunnel drying, vacuum drying, drum drying.

According to a second aspect of the invention, there is provided amethod of producing a diatom biomass, the method comprising:

-   -   a. cultivating a diatom in a culture medium to produce an active        culture;    -   b. removing a portion of the active culture to collect the        biomass;    -   c. adding fresh culture medium to the remaining active culture        that is substantially equivalent in volume to the portion of the        active culture removed in step (b) and further cultivating the        diatom; and    -   d. repeating step (b) and step (c) as desired so that at least        20 g dry cell weight of biomass per liter of active culture is        collected in a 24 hour period,    -   wherein the culture medium is designed to provide the essential        nutrients to maintain the diatom in log phase growth.

Preferably, the addition of fresh culture medium in step (c) is done inone portion.

Alternatively, the addition of fresh culture in step (c) is done inmultiple portions that total to a substantially equivalent volume to theportion removed in step (b).

Preferably the diatom produces at least one highly unsaturated fattyacid (HUFA).

Preferably the HUFA is selected from any one or more of EPA, DHA andARA.

Preferably the HUFA is an omega-3 fatty acid.

Preferably the diatom biomass contains a total HUFA level of at least 2%of dry cell weight of the biomass. Preferably the diatom biomasscontains a total HUFA level of at least 3% of dry cell weight of thebiomass.

Preferably the diatom biomass contains a mixture of EPA and DHA at alevel of at least 2% dry cell weight of the biomass.

Preferably the diatom biomass contains at least one HUFA at a level ofat least 2% of dry cell weight of the biomass.

Preferably the diatom biomass contains EPA at a level of at least 2% ofdry cell weight of the biomass.

Preferably step (b) and step (c) are repeated so that at least 25 g drycell weight of biomass per liter of active culture is collected in a 24hour period. Preferably step (b) and step (c) are repeated so that atleast 30 g dry cell weight of biomass per liter of active culture iscollected in a 24 hour period.

Preferably at least 20 g dry cell weight of biomass is collected perliter of active culture each day for at least three consecutive days.Preferably at least 20 g dry cell weight of biomass is collected perliter of active culture each day for at least seven consecutive days.Preferably at least 20 g dry cell weight of biomass is collected perliter of active culture each day for at least thirty consecutive days.Preferably at least 20 g dry cell weight of biomass is collected perliter of active culture each day for at least sixty consecutive days.

Preferably steps (b) and (c) are repeated at least 5 times. Preferablysteps (b) and (c) are repeated at least 20 times. Preferably steps (b)and (c) are repeated at least 100 times.

Preferably the density of biomass in the active culture is at least 20g/L. Preferably the density of biomass in the active culture is at least30 g/L.

Preferably the method does not include perfusion of the active culture.

Preferably the diatom is a Nitzschia species. Preferably the diatom isNitzschia laevis. Preferably the diatom is Nitzschia laevis strain In1CS20.

Preferably the diatom is a Cyclotella species.

Preferably the diatom is a Phaeodactylum species.

Preferably, the diatom is cultivated heterotrophically.

Preferably the culture medium comprises a reduced carbon source.

Preferably the reduced carbon source is selected from any one or moreof: glucose, fructose, high fructose corn syrup, monosaccharides,disaccharides, alcohols, acetic acid or its salts.

Preferably, a source of silicate is added continuously during the courseof the culture.

Preferably the source of silicate is an alkali metal silicate.Preferably the alkali metal silicate is sodium silicate or potassiumsilicate.

Preferably the sodium silicate is added continuously at a level ofbetween 20 and 120 mg sodium metasilicate pentahydrate for every gramdry cell weight of biomass produced in the culture.

Preferably the silicate is added at between 40 and 100 mg sodiummetasilicate pentahydrate per gram of dry cell weight of biomassproduced. Preferably the silicate is added at 75 mg sodium metasilicatepentahydrate per gram of dry cell weight of biomass produced.

Preferably the culture pH is maintained within the range 7.0 to 9.0.More preferably the culture pH is maintained within the range 7.5 to8.5. Most preferably the culture pH is maintained within the range 8.0to 8.5.

Preferably the culture is maintained at a temperature of between 15 and30° C. More preferably the culture is maintained at a temperature ofbetween 20 and 25° C.

Preferably agitation and/or back pressure are provided to maintain adissolved oxygen level at or above 10% of air saturation.

Preferably the dissolved oxygen level is maintained at or above 20% ofair saturation. Preferably the dissolved oxygen level is maintained ator above 30% of air saturation. Preferably the dissolved oxygen level ismaintained at or above 40% of air saturation.

Preferably steps (b) and (c) are carried out sequentially.

Alternatively steps (b) and (c) are carried out simultaneously.

Preferably the method includes the removing of a portion of the activeculture in step (b) and the adding of fresh culture medium in step (c)such that there is replacement of at least 50% of the active culturevolume every 24 hours.

Preferably the method includes the removing of a portion of the activeculture in step (b) and the adding of fresh culture medium in step (c)such that there is replacement of at least 10% of the active culturevolume every 4 hours.

Preferably the method includes the removing of a portion of the activeculture in step (b) and the adding of fresh culture medium in step (c)such that there is replacement of at least 5% of the active culturevolume every 2 hours.

Preferably the method includes removing a portion of the active culturein step (b) and the adding of fresh culture medium in step (c)continuously at a rate of at least 0.2% of the active culture volumeevery 5 minutes.

Preferably step (a) includes at least 500 liters of active culture.

Preferably the method further includes concentrating the portion of theactive culture removed in step (b), to produce a biomass concentrate.

Preferably the biomass is concentrated by any one or more of:centrifugation, flocculation, filtration, or floatation.

Preferably the biomass is concentrated by continuous centrifugation.

Preferably the method further includes the step of drying of the biomassconcentrate.

Preferably the biomass concentrate is dried by any one or more of: spraydrying, freeze drying, tunnel drying, vacuum drying, drum drying.

Alternatively the portion of the active culture removed in step (b) maybe dried directly by any one or more of: spray drying, freeze drying,tunnel drying, vacuum drying, drum drying.

According to a third aspect of the invention, there is provided anactive culture in a continuous culture, the active culture comprisingculture medium, a diatom at a diatom biomass density of at least 20 g/L.

Preferably the density of biomass is at least 30 g/L.

Preferably, the diatom produces at least one highly unsaturated fattyacid (HUFA).

Preferably the HUFA is selected from any one or more of EPA, DHA andARA.

Preferably the HUFA is an omega-3 fatty acid.

Preferably the diatom biomass contains a total HUFA level of at least 2%of dry cell weight of the biomass. Preferably the diatom biomasscontains a total HUFA level of at least 3% of dry cell weight of thebiomass.

Preferably the diatom biomass contains a mixture of EPA and DHA at alevel of at least 2% dry cell weight of the biomass.

Preferably the diatom biomass contains at least one HUFA at a level ofat least 2% of dry cell weight of the biomass.

Preferably the diatom biomass contains EPA at a level of at least 2% ofdry cell weight of the biomass.

Preferably the diatom is selected from Nitzschia species, Cyclotellaspecies, Phaeodactylum species.

Preferably the diatom is Nitzschia laevis. Preferably the diatom isNitzschia laevis strain In1CS20.

According to the fourth aspect of the invention there is provided adiatom biomass produced by the method of first or second aspects of theinvention.

According to the fifth aspect of the invention there is provided acomposition of a highly unsaturated fatty acid or an ester thereofobtained from the biomass produced by the methods of the first or secondaspects of the invention.

According to a sixth aspect of the invention there is provided a methodof producing a composition of a highly unsaturated fatty acid (HUFA) oran ester thereof, the method including the step of extracting the HUFAor an ester thereof from the biomass produced by the methods of thefirst or second aspects of the invention.

Preferably the HUFA is in its native form, being a triglyceride,phospholipid, or glycolipid. Preferably the HUFA in its native form isextracted from the biomass using a polar solvent.

Alternatively the HUFA in its native form is extracted from the biomassusing a nonpolar solvent.

Alternatively the HUFA in its native form is extracted from the biomassusing a solvent composition comprising a combination of polar andnonpolar solvents.

Preferably the polar solvent is selected from any one or more of analcohol, acetone, and polar supercritical fluids.

Preferably the non-polar solvent is selected from any one or more ofhexane, isohexane, triglycerides, and nonpolar supercritical fluids.

Preferably the extracted HUFA in its native form is subsequentlyesterified in the presence of ethanol to form a fatty acid ethyl ester(FAEE) of the HUFA.

Preferably the FAEE of the HUFA is purified by any one or a combinationof chromatography, solvent partitioning, short path distillation,molecular distillation, and High-performance liquid chromatography(HPLC).

Preferably the HUFA in its native form is saponified to produce a freefatty acid of the HUFA.

Preferably the HUFA in its free fatty acid form are purified bychromatography, molecular distillation and/or HPLC.

Alternatively the HUFA in its native form is purified by chromatography.

According to an seventh aspect of the invention there is provided anutritional supplement for humans and/or animals comprising the biomassof the third or fourth aspects of the invention.

According to an eighth aspect of the invention there is provided anutritional supplement for humans and/or animals comprising the biomassproduced by the first or second aspects of the invention.

According to a ninth aspect of the invention there is provided anutritional supplement, medical food, food or feed additive orpharmaceutical product comprising the composition produced by the methodof the sixth aspect of the invention.

Further aspects of the invention, which should be considered in all itsnovel aspects, will become apparent to those skilled in the art uponreading of the following description which provides at least one exampleof a practical application of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings in which:

FIG. 1 shows amount of biomass harvested in grams per day per liter of acontinuous culture of the invention.

FIG. 2 shows culture dry weights over time for cultures with differentprovisions of sulfur.

FIG. 3 shows graph of culture dry weights achieved against sulfurprovision.

FIG. 4 shows graph of dry cell weight at which the cultures divergedfrom log phase growth against provision of sulfur.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In general terms, the invention relates to methods of culturing a diatomto produce diatom biomass at commercially useful production rates.

Diatom biomass contains a number of products of commercial interestincluding lipids, fatty acids, amino acids, pigments and complex naturalproducts of pharmacological interest. The biomass itself also hascommercial use.

The production of highly unsaturated fatty acid (HUFA) is of particularinterest, particularly the production of the HUFA eicosapentaenoic acid(EPA). It has been found that diatoms can be induced to produce EPA inthe absence of high levels of co-concentrating compounds such as DHA,which allows for the production of EPA that is capable of being furtherconcentrated/purified.

It was previously thought that production of biomass by microbes couldonly be economic if the microbes were grown under batch or fed batchfermentation conditions where the maximum density of microbial biomassis reached. In particular, it was also believed that some classes ofmicrobes, such as diatoms, could not even be used for economicproduction of biomass under batch or fed-batch fermentation conditionsbecause the cultures suffer from slow growth and/or they have arelatively low proportion of their biomass as the desired commercialproduct, or their unique nutritional requirements make them verydifficult to grow. Diatoms have particular and unusual environmental andnutritional needs, including a requirement for large amounts of silica(a structural element of their cell walls) and, for the most part, arequirement for a saline growth medium. Whilst these fermentativemicrobes can be grown in fed-batch mode in classical fermenters in thelaboratory, the characteristics of the organisms result in such a lowvolumetric productivity that the cost of industrial scale fermentationbecomes prohibitive.

The phases of microbial growth are broadly classified as lag phase(where the microbes are adapting themselves to the growth conditions),log phase (also referred to as logarithmic phase or exponential phase,where the microbes are multiplying at an exponential rate), stationaryphase (where the maximum cell density is reached as growth rate anddeath rate are equal; this is often due to a growth limiting factor suchas deletion of nutrients) and death phase (where the death rate ishigher than the growth rate).

Surprisingly, the inventors have found it possible to achieve volumetricproductivities sufficiently high to become economic through the use ofcontinuous culture of diatoms. By maintaining the culture in, or closeto, log-phase growth these diatoms may have a lower biomass densityand/or content of the desired molecules at the time of harvest comparedto conventional stationary phase culture produced by batch and fed-batchfermentations, but the overall productivity of the culture was found tobe far higher than can be achieved in batch or fed-batch culture.Further, the productivity of desired HUFA was found to be higher.Typically, the transfer into stationary phase of a culture results inthe induction of the production of large amounts of commercially usefulproducts and the transfer into stationary phase of a microbial cultureis typically initiated by some form of nutrient limitation, typicallynitrogen limitation. The inventors, however, surprisingly found thatwith diatoms, particularly diatoms which can be grown heterotrophically,this is often not the case and nutrient limitation can result in theproduction of undesired molecules rather than the products for which theculture is being grown. The inventors surprisingly attained a muchhigher volumetric productivity of biomass and certain desired products,such as HUFAs, when the nutrients were not limited and the culture wasmaintained in a logarithmic phase of growth.

Continuous culture is where fresh culture medium is continuously added,while the active culture that is maintained in log phase growth iscontinuously removed to keep the culture volume constant.Semi-continuous culture is where a volume of the active culture (i.e.cells activity growing in log phase in a culture medium) is removed atregular or at least periodic time intervals and an equal orsubstantially similar volume (i.e. approximately equivalent) of freshmedium is added back to the culture to replace that which has beenremoved such that the average volume of the active culture remainssubstantially constant over the time of the culture. For asemi-continuous culture it is usual to first remove a portion of theactive culture prior to replacing the volume with fresh culture medium,since this sequence of events does not require additional space in thefermentor vessel. However, it is possible to reverse the sequence ofevents if there are particular commercial benefits for doing so. For theavoidance of doubt unless the context clearly requires otherwise,throughout the description and the claims, reference to “continuousculture” or “continuously culturing or the like” should be taken toinclude both continuous culture and semi-continuous culture.

A person skilled in the art would be aware that the culture medium willrequire nutrients to allow and sustain the growth of the microbe. Theculture medium will comprises a nitrogen source, (for example but notlimited to ammonium ion, nitrate, yeast extract, soy flour, peptone,tryptone), a phosphate source and a sulfur source. However, for growthof diatoms the culture medium also needs to contain a source of silica,since silica is the basis of the cell walls of all diatoms. The culturemedium additionally contains one or more of: magnesium, calcium, cobalt,manganese, boron, zinc, molybdenum, iron, chromium, nickel, selenium,copper and vitamins. It will be apparent to a person skilled in the artthat the nutrients in the culture medium of diatoms will besignificantly different from those of other microorganisms. However,when provided with the teaching in the present application a personskilled in the art would either be aware of or would be able to useexperimentation to determine the components required in the culturemedium that would not limit the growth of diatoms.

However, surprisingly, the inventors have found that there are keydifferences in the way in which a medium needs to be designed to supportcontinuous culture. Traditionally, limitation in a single nutrient canbe used to determine how much of this particular nutrient is required tomake a certain dry weight of biomass. For continuous culture, however,the inventors have found that the medium is preferably designed so thatthe active culture remains in log-phase. The inventors have found thatthis results in higher nutrient requirements on a per gram dry cellweight basis. In the methods of the invention, the culture mediumpreferably is designed to provide all the essential nutrients inquantities to maintain the diatoms at, or near log phase growth.

Surprisingly, the inventors have found it is not sufficient to simplytake processes that operate at low culture densities and scale theconcentration of nutrients in the culture medium to make more biomass;particularly careful attention must be given to culture medium design.The inventors have found that at low culture densities, imbalances innutrients are relatively unimportant since the concentrations ofcompounds are generally low. However, for fast, continuous growth athigh biomass density (as shown in the present invention), nutrientprovision must be closely matched to the needs of the organism whilegrowing in the logarithmic phase. To achieve high culture densities,relatively high concentrations of nutrients are required, but theinventors have also found that nutrients provided in excess to diatomscan also accumulate over time to levels at which they alter biomasscomposition and/or inhibit growth rate. Certain cellular requirementssuch as zinc or copper can actually be toxic to the diatoms whenprovided in too high a concentration. Addition of copper salts, forexample, is a well-established method of killing diatoms and preventingtheir regrowth in ponds, lagoons and pools. Diatoms appear to beunexpectedly susceptible to overprovision of a number of nutrients thathave toxic effects at high concentration.

Nutrients that are under-provided become depleted over the course ofseveral consecutive harvests, again resulting in undesirable changes inbiomass composition or alterations in growth. The inventors have foundthat whilst in certain circumstances, slight limitations of one or morenutrients may be desirable, for example if it results in a greaterproportion of HUFA in the biomass, in general the reduction of growthrate (i.e., transfer into a stationary phase) is undesirable. As aresult, the inventors have found that there must be particular attentionpaid to elements of the medium such as trace metals and phosphate forwhich the diatoms may have significant internal stores, since depletionof these will occur over a longer time frame.

Even if one has achieved the correct nutrient balance at one particularlevel of biomass productivity, one cannot simply double the level of allingredients in the medium and expect double the level of biomassproductivity. Addition of some ingredients, such as a reduced carbonsource, may be directly related to the amount of biomass that isproduced, but can also have an inhibitory effect on growth at higherconcentrations. As a result, feeding strategies need to be evolved toameliorate this fundamental problem. For example, it may be preferableto provide replacement culture medium for diatoms in discrete portionsover time rather than replace the whole volume of harvest at one time.Some ingredients such as sodium chloride, for example, are present forthe osmotic balance of the medium and are therefore not provideddirectly in proportion to the biomass produced. Some nutrients, such aszinc, must be maintained within a certain range of concentrations. Toolow and growth rates drop, too high and toxic effects cause reducedgrowth and eventually culture death. Some nutrients, such as potassium,play dual roles and so a balance has to be made between, for example,their osmotic effect, and use as a nutrient. While the quantities ofnutrients in the culture will be dependent on the microbe used and thevolume of culture, after being provided with the insight and teaching inthe present application that it is desirous to maintain diatom cells inlog phase growth in continuous culture, and that this requires adifferent media composition than is required for normal fermentations, aperson skilled in the art would be able to use routine experimentationto determine the quantities of components required in the culture mediumto maintain the diatom at, or near log phase growth. For example, theinventors have shown in Example 8 an example of a procedure to determinethe quantity of sulfur required for maintaining an active, log-phasegrowth of a diatom.

Of particular issue when dealing with culture of diatoms is theprovision of silicate. Even at relatively modest concentrations, theinventors have found that silicate can interact with other mediumingredients to form precipitates and gels, removing both itself andother nutrients from solution and making them unavailable for use by thediatoms. As the amount of biomass being produced increases, specialattention needs to be paid to the addition of the silicate (which isprovided proportionally to the biomass to be produced), to prevent thisfrom occurring. The silicate is preferably added continuously during thecourse of the culture to aid in the reduction of silicate precipitatesforming, and to aid in providing a quantity of silicate to maintain theculture at, or near log phase growth, (for example a method of additionof silicate is described in WO2012/053912). The source of silicate ispreferably an alkali metal silicate, for example (but not limited to)sodium silicate or potassium silicate. In preferred embodiments, sodiumsilicate is added continuously at a level of between 20 and 120 mgsodium metasilicate pentahydrate for every gram dry cell weight ofbiomass produced in the culture, more preferably between 40 and 100 mgsodium metasilicate pentahydrate per gram of dry cell weight of biomassproduced, and most preferably 75 mg sodium metasilicate pentahydrate forevery gram dry cell weight of biomass produced in the culture.

The inventors have found that it is possible to continuously culturediatoms at high densities, in the region of 20 g Dry Cell Weight (DCW)/Lor higher, for example 20 to 150 g DCW/L, or more preferably 30 to 100 gDCW/L, even more preferably 30 to 70 g DCW/L. Preferably the dry cellweight of the culture at the time of harvest remains within +/−5% of themean of harvests during the time over which the culture runs in order tomaintain a high density of diatoms in the culture. Such densities ofdiatoms in the continuous culture of the invention allow for highvolumetric production rates of at least 20 grams DCW of biomass perliter of active culture per day (DCW/L/day) (i.e. 20 grams of biomassproduced per liter of active culture in the fermentation vessel perday), more preferably at least 30 grams DCW of biomass per liter ofactive culture per day, but may be optimized using the teaching of thepresent application to be as high as 50 g DCW/L/day of biomass, morepreferably 60 g DCW/L/day of biomass, even more preferably 70 gDCW/L/day of biomass or higher. The range of the production rates istherefore any one of the preferred minimum values to any one of thepreferred maximum values, for example (but not limited to), 20 to 50 gDCW/L/day of biomass, more preferably 20 to 60 g DCW/L/day of biomass,more preferably 20 to 70 g DCW/L/day of biomass.

The continuous cultures of the methods of the invention preferablyinclude replacement of at least 35% by volume of the active cultureevery 24 hours, more preferably at least 40% by volume, more preferablyat least 50% by volume, even more preferably at least 60% by volume. Themethods alternatively include replacement of at least 6% by volume ofthe active culture every 4 hours, more preferably at least 8% by volume,more preferably at least 10% by volume, more preferably at least 12% byvolume most preferably at least 15% by volume. In a further alternativethe methods of the invention preferably include replacement of at least3% by volume of the active culture every 2 hours, more preferably atleast 4% by volume, more preferable at least 6% by volume, mostpreferably at least 7% by volume. In yet a further alternative, themethods of the invention preferably include replacement of at least0.15% by volume of the active culture every 5 minutes, more preferably0.2% by volume, more preferably at least 0.3%, most preferably at least0.35% by volume. The volume of harvest and replacement should be chosento match the growth rate of the organism in log phase so that theculture density in sequential harvests does not drop and the culturedoes not enter stationary phase. Whilst semi-continuous harvest every24, 4, and 2 hours and continuous harvest are provided by way ofexamples, any interval of less than 24 hours that allows the cells toremain in logarithmic growth may be used so that the interval betweenharvests can be chosen to suit the needs of downstream processingalthough it is noted that, for a given dry weight at harvest and growthrate, more frequent harvests provide a higher biomass productivity.

In some cases, previous culture methods have used perfusion to increasethe density and productivity of cultures of microbes. Perfusion is wherecells and medium from active culture are separated, cells are returnedto the fermenter and additional fresh medium is added. The methods ofthe present invention preferably do not include perfusion of the activeculture, as this procedure adds further costs and procedures which wouldbe impractical on an industrial scale for biomass production. Perfusioncultures are also extremely susceptible to contamination and requireextraordinary measures to maintain axenic cultures for long periods oftime. The maintenance of sterility is important since invadingcontaminant organisms that grow faster than the production organism canquickly come to dominate the culture over the course of several harvestsat which point the culture must be abandoned. The methods of theinvention surprisingly provide high productivity culture, without theneed for perfusion. While the inventors have found care should be takento keep contaminants out of the fermenter during the methods of theinvention particularly at both the harvest of culture and refill offresh medium, this problem is lessened by the lack of need forperfusion.

The inventors have shown that the methods of the invention can becarried out at scales of at least 12 liters of active culture, morepreferably at least 14 liters of active culture, more preferably atleast 400 liters of active culture, even more preferably at least 500liters of active culture. However, once provided with the teaching inthe present application larger scale cultures such as at least 10,000liters, at least 100,000 liters, and even at least 200,000 liters can beused for industrial production.

To be commercially viable, the continuous cultures of the invention arepreferably sustainable over a period of time. The inventors havedemonstrated the methods of the invention can be maintained for at least72 hours (3 days), more preferably at least 5 days, even more preferablya week (7 days), even more preferably at least a month (30 days), mostpreferably at least 2 months (60 days). For a semi-continuous culturethe cycle of removal of a portion of active culture and the replacementof fresh culture medium is preferably repeated at least 5 times.However, in the examples of the invention provided in the presentapplication (see Examples 1-5), the cultures were discontinued after 60days (after the removal of active culture and replacement with freshmedium well over 100 times) without any indication that the culturescould not be maintained longer. Thus, there is no indication thecultures could not be continued for periods of months or years withoutissue. In a preferred embodiment the average volumetric production rateof biomass is at least 20 g dry weight/L/day over a period of at least72 hours (3 days), more preferably at least a week (7 days), morepreferably at least a month (30 days), even more preferably at least 2months (60 days).

Preferably the methods of the invention comprise cultivating anidentified diatom. Preferably the strain of diatom is selected, whenunder culture conditions, for a capability to produce at least one HUFA,preferably EPA and/or DHA. A person skilled in the art will be aware ofdiatom species that are capable of producing HUFA, or would be able tocarry out routine experimentation in order to determine this (forexample, see Dunstan et al. (1993) Phytochemistry 35:155-161). Inpreferred embodiments, the diatom is selected from, but not limited, toa Nitzschia species, a Cyclotella species or a Phaeodactylum species.Most preferably the diatom is Nitzschia laevis, for example (but notlimited to) Nitzschia laevis strain In1CS20.

A person skilled in the art would be aware of methods to “seed” thefermentation with the required microbe. The fermentation will generallybe carried out in a fermentation vessel, or other suitable container aswould be apparent to a person skilled in the art.

The diatom biomass produced by the methods of the present invention isin a metabolic state (log-growth phase) that is quite different incomposition from stationary phase cultures. When diatoms which arecapable of producing highly unsaturated fatty acids (HUFA) are used,these conditions result in relatively high levels of one or more HUFA.Most preferably the HUFA is EPA, but it may also beneficially be amixture of more than one HUFA, preferably EPA, DHA and ARA, morepreferably EPA and DHA. Both EPA and DHA are omega-3 fatty acids and areknown to have particularly beneficial health properties. These fattyacids, or their esterified forms, can be used as pharmaceuticals,medical foods, food or feed additives, cosmetic products, or nutritionalsupplements. ARA is an omega-6 fatty acid and is also known to havebeneficial health properties. Preferably the diatom biomass contains atotal HUFA level (which can be made up of a mixture of different HUFA)of at least 2% of dry cell weight of the biomass, more preferably atleast 3% of dry cell weight of the biomass. More preferably the diatombiomass contains a mixture of EPA and DHA at a level of at least 2% drycell weight of the biomass, even more preferably at least 3% of dry cellweight of the biomass. At least one HUFA is preferably produced by themethods of the invention at a level of at least 2% of dry cell weight ofthe biomass (i.e. a single HUFA at a level of 2%), more preferably atleast 2.5% of dry cell weight of the biomass, more preferably at least2.7% of dry cell weight of the biomass, even more preferably at least3.2% of dry cell weight of the biomass. The HUFA produced is mostpreferably EPA. The maximum level of total HUFA or single HUFA in thebiomass will be dependent on the fine tuning of conditions and thechoice of diatom, once a person skilled in the art is provided with theteaching in the specification. However, the inventors believe that themaximum limit is likely to be in the region of 5%, more preferably 10%,more preferably 15%, even more preferably 20% of dry cell weight. Therange of the content of one or more HUFA in the biomass is therefore anyone of the preferred minimum values to anyone of the preferred maximumvalues, for example (but not limited to), the level of HUFA ispreferably 1 to 20% of dry cell weight of the biomass, more preferably 2to 15% of dry cell weight of the biomass, more preferably 2 to 10% ofdry cell weight of the biomass, more preferably 2.7 to 10% of dry cellweight of the biomass, even more preferably 3.2 to 10% of dry cellweight of the biomass.

Current art teaches that most microalgae must be culturedphotosynthetically to produce HUFA. In a particularly preferred aspectof the invention, the inventors have surprisingly found that diatoms areable to be cultured heterotrophically using continuous fermentation toproduce HUFA at a high productivity. This allows for commercialproduction without the need to provide light for the high densityfermentation, thus overcoming the engineering issues associated withproviding sufficient light to high density cultures previouslydiscussed. Where the diatoms are cultivated heterotrophically, a reducedcarbon source is preferably provided in the culture medium. Preferablythe reduced carbon source is selected from (but not limited to) any oneor more of: glucose, fructose, high fructose corn syrup,monosaccharides, disaccharides, alcohols, acetic acid or its salts.

Current art teaches that, to gain high volumetric productivity of HUFAs,batch or fed-batch conditions should be used where cells are taken intostationary phase in which a state of oleogenesis is induced, usuallythrough the use of nitrogen and/or phosphate limitation and thecomposition of the algal biomass is thereby significantly different fromcompositions of biomass growing in log-phase. In a particularlypreferred aspect of the invention, the inventors have surprisinglydetermined that diatoms can be cultured heterotrophically usingcontinuous fermentation of the diatom maintained in log-phase to produceHUFA at high volumetric productivity. This allows for commercialproduction of organisms, such as diatoms, which are unsuited to batch orfed-batch fermentation, thus overcoming the issues with growing them atscale.

The inventors have also found the culture pH is preferably maintainedwithin the range 7.0 to 9.0, more preferably within the range 7.5 to8.5, most preferably within the range 8.0 to 8.5. The temperature of theculture is preferably maintained between 15 and 30° C., more preferablyat a temperature of between 20 and 25° C. Preferably agitation and/orback pressure are provided to maintain a dissolved oxygen level at orabove 10%, more preferably 20%, even more preferably 30% of airsaturation. The dissolved oxygen level is even more preferablymaintained at or above 40% of air saturation. Such conditions promotegrowth of the diatom and HUFA formation (when a diatom capable ofproducing HUFA is used). The dissolved oxygen levels are preferablymaintained for at least 80%, more preferably 90% of the cultivationtime. Brief drops below the preferred dissolved oxygen levels areconsidered within the scope of the present invention as they should notsignificantly affect the culture.

The invention also includes the active culture (i.e. diatom cellsactivity growing in log phase in a culture medium) in the continuousculture which, prior to separation of the biomass, extraction orpurification steps, will include the culture medium, diatoms at abiomass density of at least 20 g/L. Preferably, where a diatom capableof producing HUFA is used, the diatom biomass contains a highlyunsaturated fatty acid (HUFA).

Following separation of portions of the active culture from thecontinuous culture, the diatom biomass component is preferablyconcentrated to substantially remove or separate the liquid component toproduce a biomass concentrate, for example by centrifugation orcontinuous centrifugation or other means known in the art for harvestingbiomass, for example but not limited to centrifugation, flocculation,filtration and/or floatation. The cells of the biomass can alsooptionally be washed to remove excess medium and/or killed by heattreatment (e.g., pasteurization) or otherwise (for example to denatureendogenous enzymes which may decrease product yield). The biomass isoptionally dried to reduce or eliminate water, for example by spraydrying, freeze drying, tunnel drying, vacuum drying and/or drum drying.The dried biomass is optionally milled to form a fine powder, oroptionally formed into pellets. Alternatively the portion of activeculture removed from the continuous culture may be dried directlywithout pre-concentration.

The diatom biomass of the invention may be subjected to one or moreextraction steps to extract desired products, for example HUFA or estersthereof, from the biomass to yield a composition (for example a HUFAcomposition). Suitable extraction techniques are well known in the artand will be dependent on the desired product. For example to extract aHUFA, the biomass may be extracted with a non-selective lipid solvent(e.g. near critical di-methyl ether or ethanol) and recovered from thesolvent as a residue. Alternatively, the biomass may be extracted usingother polar or nonpolar solvents including, but not limited to hexane,alcohol, acetone, supercritical or liquid carbon dioxide, or mixturesthereof (for example hexane and isopropanol). The biomass may beextracted with solvent either in a batch or a counter-current fashion.The solvent (containing the HUFA) is separated from the extractedbiomass, for example by (but not limited to) settling, filtration,centrifugation. The HUFA (or ester thereof) are recovered from thesolvent.

The HUFA present in the biomass are in the form of free fatty acidsand/or esterified forms of the fatty acid. Examples of esterified formsof the fatty acid include triglycerides, phospholipids, and glycolipids(collectively referred to as lipids). The lipids are the native form ofthe HUFA, (i.e. the form naturally found in the biomass before anyexternal chemical modification). The free fatty acids are either cleavedfrom the lipids while still in the biomass (for example by the action ofenzymes), or can optionally by cleaved from the lipids (for example toform an alternative ester, or to the free fatty acid) during theextraction, enriching and/or purification steps.

Following extraction, the further step of enriching HUFA or purifyingone or more HUFA from the mixture may be performed using techniques wellknown in the art. For example, the extracted material may be treatedwith acids, alkalis and/or enzymes in the presence of alcohol or waterto form free fatty acids or fatty acid alkyl esters. For example, theHUFA may be transesterified to form the fatty acid ethyl ester (FAEE).The ester may optionally be purified in order to achieve a requiredstandard of purity. Examples of purification processes includechromatography, molecular distillation and/or high-performance liquidchromatography (HPLC). For the avoidance of doubt HPLC is sometimesreferred to as high-pressure liquid chromatography. Alternatively thelipids in the biomass are saponified to form the free fatty acids. Thefree fatty acids are optionally purified or separated, for example, by(but not limited to) chromatography. The HUFA, purified in the freefatty acid form, may then be used as such or converted into an ethylester (FAEE) using processes well known in the art.

Following purification of the HUFA, or ester thereof, the HUFA can thenbe used for various food or feed applications suitable for human and/oranimal consumption including, but not limited to, pharmaceuticalproducts, medical foods, food additives, cosmetic products, dietarysupplements, or feed additives. In some cases the biomass itself can beused for various food or feed applications including, but not limitedto, pharmaceutical products, medical foods, food additives, cosmeticproducts, dietary supplements, or feed additives.

By using the methods of the invention to provide controlled cultivationof a diatom, a HUFA product can be provided in a consistent, sustainableand traceable form (i.e., by good manufacturing processes) without theconcern for environmental pollutants, all of which plague fish oil as asource for similar HUFA products.

Definitions and Abbreviations

Highly unsaturated fatty acid (HUFA) is a fatty acid containing 20carbons or more, with 4 or more double bonds. They may be omega-3 oromega-6.

Fatty acids are described in the form CX:Y, wherein the number Xdescribes the number of carbon atoms and the number Y describes thenumber of double bonds in the fatty acid. Where Y equals zero the fattyacid is described as saturated, where Y is greater than zero the fattyacid is described as unsaturated. The position and type of the doublebonds may be specified as, for example, “cis 5, 11, 14” where thenumbers reflect the location of the carbon-carbon double bonds, countingfrom the carboxylic acid end of the molecule.

Unless the context clearly requires otherwise, throughout thedescription and the claims, reference to “fatty acid” should be taken toinclude both the free fatty acids and esterified forms of the fatty acidwhich are suitable for human use (e.g., for consumption or topicalapplications), Examples of esterified forms of the fatty acid which aresuitable for human consumption include triglycerides, phospholipids,glycolipids, and ethyl esters. Methyl esters are unfavorable incompositions for human consumption because they release methanol intothe body during processing in the human gut and they are therefore arenot preferred within the compositions and methods for production. A termsuch as C20:5, for example, is understood to include both the free fattyacid and esterified forms of the fatty acid (not including methylesters) with the number of carbon atoms and double bonds referringsolely to the fatty acid portion of the ester.

Omega-3 fatty acid is a fatty acid with the first double bond threecarbon atoms from the methyl end (the omega end) of the molecule.Omega-3 is often shortened to n−3 and both terms are herein usedinterchangeably.

Omega-6 fatty acid is a fatty acid with the first double bond six carbonatoms from the methyl end of the molecule. Omega-6 is often shortened ton−6.

EPA, C20:5 n−3, Eicosapentaenoic acid, is an omega-3 fatty acid withtwenty carbon atoms and five double bonds.

ARA, C20:4 n−6, Arachidonic acid, is an omega-6 fatty acid with twentycarbon atoms and four double bonds.

DHA, C22:6 n−3, Docosahexaenoic acid, is an omega-3 fatty acid withtwenty-two carbon atoms and six double bonds.

DCW, dry cell weight, means the weight of a biomass once all water hasbeen removed.

Log phase or logarithmic growth is used synonymously with exponentialgrowth throughout this description and all are used consistently withdefinitions well known in the art.

Heterotrophic culture means a culture of organisms for which at least90% of the energy supply for the culture is derived from suppliednutrients which are usually a form or forms of organic carbon (e.g.glucose, acetate). Therefore a maximum of 10% of the energy supply maybe derived from light energy. Preferably, less than 5% or morepreferably less than 1% of the energy supply is derived from lightenergy. More preferably, the whole of the energy supply is from suppliednutrients.

Active culture means a biomass of cells activity growing in log phase ina culture medium contained in a suitable vessel, for example afermenter.

Photoautotrophic culture (or photoautotroph microbes or diatoms) means aculture of organisms for which the sole energy source is light.

Nutrient limitation means that the absence or low level of the nutrientin question causes the organism to undergo metabolic changes that wouldnot occur if the nutrient were present at higher levels and isessentially the entrance into the stationary phase of a culture. Aculture would be considered to be in a non-limiting nutrient conditionif growth proceeds in a logarithmic fashion.

Unless the context clearly requires otherwise, throughout thedescription and the claims, reference to “continuous culture” or“continuously culturing” should be taken to include both strictcontinuous culture (where fresh culture medium is continuously added,while active culture is continuously removed to keep the culture volumeconstant) and semi-continuous culture (where a volume of active cultureis removed at regular or at least periodic time intervals and an equalor substantially similar volume of fresh medium is added to the cultureto replace that which has been removed such that the average volume ofactive culture remains substantially constant over the time of theculture). For a semi-continuous culture it is usual to first remove aportion of the active culture prior to replacing the volume with freshculture medium, since this sequence of events does not requireadditional space in the fermentor vessel. However, it is possible toreverse the sequence of events.

Unless the context clearly requires otherwise, throughout thedescription and the claims, reference to “diatoms” or “diatom” should betaken to include all organisms of the class Bacillariophyceae includingbut not limited to pennate (e.g., Nitzschia) and centric (e.g.,Cyclotella) diatoms.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise”, “comprising”, and thelike, are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense, that is to say, in the sense of“including, but not limited to”.

It is intended that reference to a range of numbers disclosed herein(for example, 1 to 10) also incorporates reference to all rationalnumbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5,7, 8, 9 and 10) and also any range of rational numbers within that range(for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).

EXAMPLES Example 1—Semi-Continuous Culture of Nitzschia laevis

A culture of Nitzschia laevis strain In1CS20 was grown in 14 L culturein a 20 L stirred vessel. The pH was maintained at 8.0 or above by theaddition of NaOH, temperature was maintained at 20° C., and pressuremaintained at around 500 mbar above surroundings. Agitation was providedto keep dissolved oxygen above 40% saturation.

The cells were grown up to a density of around 40 g dry cell weight perliter of culture and harvest initiated. Every 4 hours, 1.4 liters ofculture was withdrawn from the vessel as harvest, and the volume wasmade back up to 14 L with hourly feeds of approximately 400 mL freshmedium. This was sustained for 7 days.

The fresh medium was composed of:

g/L Glucose monohydrate 95 Sodium Nitrate NaNO₃ 10.1 Yeast Extract 3.1Na₂HPO₄ 1.16 Sodium chloride NaCl 8.4 Potassium chloride KCl 1.17Magnesium sulfate heptahydrate MgSO₄•7H₂O 4.9 Calcium chloride dihydrateCaCl₂•2H₂O 0.204 Sodium sulphate 0.586 mg/L Cobalt chloride hexahydrateCoCl₂•6H₂O 0.081 Manganese chloride 4-hydrate MnCl₂•4H₂O 0.843 Boricacid H₃BO₃ 103.1 Zinc chloride ZnCl₂ 1.05 Sodium molybdate dihydrateNa₂MoO₄•2H₂O 1.363 Ferric chloride hexahydrate 7.22 Citrate 675 Coppersulfate 0.024 Vitamin B12 202.7 Thiamine 0.34 Biotin 0.34

In addition to the hourly feeds, sodium metasilicate was also providedto the culture as a separate, near continuous, feed such that it wasprovided at around 7.5% w/w sodium metasilicate (pentahydrate)/dry cellweight.

Optical density of the culture was recorded every 10 minutes andremained within a range +/−2% of the average throughout the 7 dayperiod. The culture dry cell weight (DCW) was measured from anadditional 100 mL sample taken at the time of harvest one on four of theseven days. This sample was also used for composition analysis.

The dry cell weight measurements were then used to calculate the DCW ateach of the other harvests.

By multiplying the volume of harvest by the DCW, a biomass collectionand thus a biomass productivity were calculated.

In the Table 1 below DCW and EPA content of biomass figures in italicsare calculated from the surrounding data.

TABLE 1 Day 1 Biomass DCW (g/L) Harvest (L) Collected (g) Harvest 1 42.01.5 63.0 Harvest 2 41.6 1.4 58.2 Harvest 3 41.4 1.4 58.0 Harvest 4 41.31.4 57.9 Harvest 5 41.2 1.4 57.7 Harvest 6 41.1 1.4 57.6 BiomassProductivity (g/L/day) 25.2 EPA Content of Biomass (%) 2.5 EPAProductivity (mg/L/h) 26.2 Day 2 Biomass DCW (g/L) Harvest (L) Collected(g) Harvest 1 41.0 1.4 57.4 Harvest 2 40.8 1.4 57.2 Harvest 3 40.7 1.456.9 Harvest 4 40.5 1.4 56.7 Harvest 5 40.3 1.4 56.5 Harvest 6 40.2 1.456.2 Biomass Productivity (g/L/day) 24.3 EPA Content of Biomass (%) 2.5EPA Productivity (mg/L/h) 25.4 Day 3 Biomass DCW (g/L) Harvest (L)Collected (g) Harvest 1 40.0 1.5 60.0 Harvest 2 39.7 1.4 55.5 Harvest 339.6 1.4 55.5 Harvest 4 39.6 1.4 55.4 Harvest 5 39.6 1.4 55.4 Harvest 639.5 1.4 55.3 Biomass Productivity (g/L/day) 24.1 EPA Content of Biomass(%) 2.5 EPA Productivity (mg/L/h) 25.1 Day 4 Biomass DCW (g/L) Harvest(L) Collected (g) Harvest 1 39.5 1.5 59.3 Harvest 2 39.3 1.4 55.1Harvest 3 39.5 1.4 55.3 Harvest 4 39.6 1.4 55.5 Harvest 5 39.8 1.4 55.7Harvest 6 39.9 1.4 55.9 Biomass Productivity (g/L/day) 24.1 EPA Contentof Biomass (%) 2.5 EPA Productivity (mg/L/h) 25.1 Day 5 Biomass DCW(g/L) Harvest (L) Collected (g) Harvest 1 40.1 1.4 56.1 Harvest 2 40.31.4 56.4 Harvest 3 40.5 1.4 56.7 Harvest 4 40.7 1.4 57.0 Harvest 5 40.91.4 57.3 Harvest 6 41.1 1.4 57.5 Biomass Productivity (g/L/day) 24.4 EPAContent of Biomass (%) 2.5 EPA Productivity (mg/L/h) 25.4 Day 6 BiomassDCW (g/L) Harvest (L) Collected (g) Harvest 1 41.3 1.4 57.8 Harvest 241.5 1.4 58.1 Harvest 3 41.7 1.4 58.3 Harvest 4 41.8 1.4 58.6 Harvest 542.0 1.4 58.8 Harvest 6 42.2 1.4 59.1 Biomass Productivity (g/L/day)25.1 EPA Content of Biomass (%) 2.5 EPA Productivity (mg/L/h) 26.1 Day 7Biomass DCW (g/L) Harvest (L) Collected (g) Harvest 1 42.4 1.5 63.6Harvest 2 41.9 1.4 58.7 Harvest 3 41.8 1.4 58.5 Harvest 4 41.7 1.4 58.4Harvest 5 41.6 1.4 58.2 Harvest 6 41.4 1.4 58.0 Biomass Productivity(g/L/day) 25.4 EPA Content of Biomass (%) 2.5 EPA Productivity (mg/L/h)26.4

Example 2

The culture of Example 1 was reset by harvesting 50% of the volume andallowing growth back to a density of around 40 g dry cell weight perliter of culture before harvest was again initiated.

For this example harvests of between 0.7 and 0.8 L were carried outevery two hours (harvest volume being adjusted to keep the dry weight atharvest close to constant). The volume was then made back up to 14 Lwith two refills; one of approximately 400 mL fresh medium immediatelyfollowing harvest and one providing the remaining volume to reach 14 Lan hour later. Medium composition was as for Example 1. Silicate was fedto the culture in the same manner as was used in Example 1.

The optical density of the culture was recorded every 10 minutes andremained within a range of +/−2% of the average throughout the 7 dayperiod. An additional 100 mL sample was taken for determination ofculture dry cell weight and biomass composition at the time of harveston six of the eight days.

Biomass Productivity was calculated as per Example 1. In the Table 2below DCW and EPA content of biomass figures in italics were calculatedfrom the surrounding data.

TABLE 2 Day 1 Biomass DCW (g/L) Harvest (L) Collected (g) Harvest 1 40.00.8 32.0 Harvest 2 39.7 0.7 27.8 Harvest 3 39.7 0.7 27.8 Harvest 4 39.70.7 27.8 Harvest 5 39.7 0.7 27.8 Harvest 6 39.7 0.7 27.8 Harvest 7 39.70.7 27.8 Harvest 8 39.8 0.7 27.8 Harvest 9 39.8 0.7 27.8 Harvest 10 39.80.7 27.8 Harvest 11 39.8 0.7 27.8 Harvest 12 39.8 0.7 27.9 BiomassProductivity 24.1 (g/L/day) EPA Content of Biomass 2.6 (%) EPAProductivity 26.2 (mg/L/h) Day 2 Biomass DCW (g/L) Harvest (L) Collected(g) Harvest 1 39.8 0.9 35.8 Harvest 2 39.4 0.8 31.6 Harvest 3 39.4 0.831.5 Harvest 4 39.3 0.8 31.5 Harvest 5 39.3 0.8 31.4 Harvest 6 39.2 0.831.4 Harvest 7 39.1 0.8 31.3 Harvest 8 39.1 0.8 31.3 Harvest 9 39.0 0.831.2 Harvest 10 39.0 0.8 31.2 Harvest 11 38.9 0.8 31.1 Harvest 12 38.90.8 31.1 Biomass Productivity 27.2 (g/L/day) EPA Content of Biomass 2.5(%) EPA Productivity 28.3 (mg/L/h) Day 3 Biomass DCW (g/L) Harvest (L)Collected (g) Harvest 1 38.8 0.9 34.9 Harvest 2 38.5 0.8 30.8 Harvest 338.5 0.8 30.8 Harvest 4 38.6 0.8 30.8 Harvest 5 38.6 0.8 30.9 Harvest 638.6 0.8 30.9 Harvest 7 38.6 0.8 30.9 Harvest 8 38.6 0.8 30.9 Harvest 938.6 0.8 30.9 Harvest 10 38.7 0.8 30.9 Harvest 11 38.7 0.8 30.9 Harvest12 38.7 0.8 30.9 Biomass Productivity 26.8 (g/L/day) EPA Content ofBiomass 2.6 (%) EPA Productivity 29.0 (mg/L/h) Day 4 Biomass DCW (g/L)Harvest (L) Collected (g) Harvest 1 38.7 0.8 31.0 Harvest 2 38.7 0.831.0 Harvest 3 38.7 0.8 31.0 Harvest 4 38.8 0.8 31.0 Harvest 5 38.8 0.831.0 Harvest 6 38.8 0.8 31.1 Harvest 7 38.8 0.8 31.1 Harvest 8 38.9 0.831.1 Harvest 9 38.9 0.8 31.1 Harvest 10 38.9 0.8 31.1 Harvest 11 38.90.8 31.2 Harvest 12 39.0 0.8 31.2 Biomass Productivity 26.6 (g/L/day)EPA Content of Biomass 2.5 (%) EPA Productivity 27.7 (mg/L/h) Day 5Biomass DCW (g/L) Harvest (L) Collected (g) Harvest 1 39.0 0.8 31.2Harvest 2 39.0 0.8 31.2 Harvest 3 39.0 0.8 31.2 Harvest 4 39.0 0.8 31.2Harvest 5 39.1 0.8 31.3 Harvest 6 39.1 0.8 31.3 Harvest 7 39.1 0.8 31.3Harvest 8 39.1 0.8 31.3 Harvest 9 39.1 0.8 31.3 Harvest 10 39.1 0.8 31.3Harvest 11 39.2 0.8 31.3 Harvest 12 39.2 0.8 31.3 Biomass Productivity26.8 (g/L/day) EPA Content of Biomass 2.4 (%) EPA Productivity 26.8(mg/L/h) Day 6 Biomass DCW (g/L) Harvest (L) Collected (g) Harvest 139.2 0.9 35.3 Harvest 2 39.0 0.8 31.2 Harvest 3 39.2 0.8 31.3 Harvest 439.3 0.8 31.5 Harvest 5 39.5 0.8 31.6 Harvest 6 39.6 0.8 31.7 Harvest 739.7 0.8 31.8 Harvest 8 39.9 0.8 31.9 Harvest 9 40.0 0.8 32.0 Harvest 1040.2 0.8 32.1 Harvest 11 40.3 0.8 32.2 Harvest 12 40.5 0.8 32.4 BiomassProductivity 27.5 (g/L/day) EPA Content of Biomass 2.3 (%) EPAProductivity 26.4 (mg/L/h) Day 7 Biomass DCW (g/L) Harvest (L) Collected(g) Harvest 1 40.6 0.85 34.5 Harvest 2 40.2 0.75 30.2 Harvest 3 40.10.75 30.1 Harvest 4 40.0 0.75 30.0 Harvest 5 39.9 0.75 29.9 Harvest 639.8 0.75 29.9 Harvest 7 39.7 0.75 29.8 Harvest 8 39.7 0.75 29.7 Harvest9 39.6 0.75 29.7 Harvest 10 39.5 0.75 29.6 Harvest 11 39.4 0.75 29.5Harvest 12 39.3 0.75 29.5 Biomass Productivity 25.9 (g/L/day) EPAContent of Biomass 2.4 (%) EPA Productivity 25.9 (mg/L/h) Day 8 BiomassDCW (g/L) Harvest (L) Collected (g) Harvest 1 39.2 0.85 33.3 Harvest 239.1 0.75 29.3 Harvest 3 39.3 0.75 29.5 Harvest 4 39.5 0.75 29.6 Harvest5 39.7 0.75 29.8 Harvest 6 39.9 0.75 29.9 Harvest 7 40.1 0.75 30.1Harvest 8 40.3 0.75 30.3 Harvest 9 40.6 0.75 30.4 Harvest 10 40.8 0.7530.6 Harvest 11 41.0 0.75 30.7 Harvest 12 41.2 0.75 30.9 BiomassProductivity 26.0 (g/L/day) EPA Content of Biomass 2.5 (%) EPAProductivity 27.1 (mg/L/h)

Example 3

The culture of Example 2 was switched from a harvest every 2 hours to aharvest once per 24 hours. At the time of harvest, 7 L (50%) of thevolume of the culture in the fermenter was removed from the fermenterover the course of 70 minutes. On completion of harvest, refill of thefermenter was started with approximately 400 mL fresh medium being addedback to the fermenter every hour until the full volume of 14 L wasreached.

Fresh medium and silicate provision were as for the previous examples.

A 100 mL sample was taken at harvest time for determination of culturedry cell weight and composition. Cell dry weights ranged from 40.4 g/Lon day 1 to 44.6 g/L on day 5 and 43.9 g/L on day 6 giving biomassproductivities of between 20.5 and 22.6 g/L/day.

EPA content of biomass was between 2.5 and 2.7% of dry cell weight.

Example 4

The culture of Example 3 was switched from harvest every 24 hours to anear continuous culture. Once the culture had reached a dry weight ofover 40 g/L, 30 mL of medium was added every 5 minutes. Silicate wasalso added as per example 1. Harvests were carried out as required tomaintain the culture volume between 14.0 and 14.2 L. Including theadditional volume added as silicate, and the volume taken as samplesapproximately 8.9 L of medium was added and harvested per day.

Samples were taken for determining culture dry cell weight andcomposition at 9 am on weekdays. Biomass productivities were calculatedon the basis that an average between two 9 am dry weight measurementswould be representative of the 24 hour period. Biomass productivitiesare shown in Table 3 below.

TABLE 3 DCW at Average dry Harvest Biomass EPA EPA 9 am weight forvolume Production Content Productivity Day (g/L) calculation (g/L) (L)(g/L) (%) (mg/L/hour) 1 44.5 42.7 8.9 27.1 2.7 30.5 2 40.9 41.2 8.9 26.22.8 30.5 3 41.7 8.9 26.5 not measured 4 42.2 8.9 26.8 not measured 542.5 42.8 8.9 27.2 2.7 30.6 6 43.1 42.9 8.9 27.3 2.9 33.0 7 42.8 42.88.9 27.2 2.8 31.8

Example 5

The culture of Example 4 was kept in continuous culture past the sevenday period of the example. After being reset with a 7.2 L harvest, itwas switched to a 4 hourly harvest regime in which the volume of activeculture removed was replaced with fresh culture medium in one additionafter harvest concluded. This continued for a further month. With theexception of day 8 of the culture, caused by the reset between Examples1 and 2, and day 18 of the culture, caused by the reset between examples2 and 3, the biomass productivity of the culture as measured from 9 amto 9 am did not drop below 20 g/L/day for a period of 60 days as shownin FIG. 1 (note that productivities over weekends after Example 4 areplotted as averages for the weekend). In the later stages of the culturebiomass productivities in excess of 35 g/L/day were seen. With EPA ataround 2.5% of biomass at this time, this gave EPA productivities ofover 35 mg EPA/L/hour. The same medium was used throughout and theincreases in productivity are attributed to changes in agitation andpressure to improve oxygen availability.

As well as producing EPA, the organism produced both ARA and DHA. Atypical biomass composition included DHA at around 0.15% of biomass andARA at around 0.1% of biomass.

Example 6

A culture of Nitzschia laevis strain In1CS20 was grown in 500 L culturein a 600 L airlift vessel. pH was maintained at 8.0 or above by theaddition of NaOH, temperature was maintained at 20° C., and pressure ataround 500 mbar above surroundings. Airflow was adjusted to keep thedissolved oxygen levels above 30% saturation at the bottom of thevessel.

The cells were grown up to a density of around 40 g dry cell weight perliter of culture and harvest initiated. Every 4 hours, 40 liters ofculture were withdrawn from the vessel as harvest and the volume madeback up to 500 L with fresh medium. This was sustained for 6 days. Onday 4, the operating volume in the fermenter was reduced to 480 sincethe airflow had risen to the point where the height of foam in thefermenter was nearing the top plate. The harvest was reducedproportionally to 38 L every 4 hours.

The fresh medium was composed of:

g/L Glucose monohydrate 120 Sodium Nitrate NaNO₃ 12.7 Yeast Extract 3.9Na₂HPO₄ 1.47 Sodium chloride NaCl 7.5 Potassium chloride KCl 1.04Magnesium sulfate heptahydrate MgSO₄•7H₂O 4.4 Calcium chloride dihydrateCaCl₂•2H₂O 0.181 Sodium sulphate 0.521 mg/L Cobalt chloride hexahydrateCoCl₂•6H₂O 0.072 Manganese chloride 4-hydrate MnCl₂•4H₂O 0.750 Boricacid H₃BO₃ 91.643 Zinc chloride ZnCl₂ 0.936 Sodium molybdate dihydrateNa₂MoO₄•2H₂O 1.212 Ferric chloride hexahydrate 6.42 Citrate 600.0 Coppersulfate 0.022 Vitamin B12 180.2 Thiamine 0.302 Biotin 0.302

In addition to the media refills, sodium metasilicate was provided tothe culture as a separate, near continuous, feed such that it wasprovided at around 7.5% w/w sodium metasilicate (pentahydrate)/dry cellweight.

The culture dry cell weight was measured at the first harvest andsamples taken at these times for composition analysis on days two tosix. Biomass productivity was calculated as per Example 1.

The culture dry weight (DCW), biomass productivity, EPA content and EPAproductivity are shown in Table 4.

TABLE 4 Day 1 Biomass DCW (g/L) Harvest (L) Collected (g) Harvest 1 46.640.0 1864.0 Harvest 2 46.6 40.0 1864.7 Harvest 3 46.6 40.0 1865.3Harvest 4 46.6 40.0 1866.0 Harvest 5 46.7 40.0 1866.7 Harvest 6 46.740.0 1867.3 Biomass Productivity (g/L/day) 22.4 EPA Content of Biomass(%) Not measured EPA Productivity (mg/L/h) unknown Day 2 Biomass DCW(g/L) Harvest (L) Collected (g) Harvest 1 46.7 40.0 1868.0 Harvest 246.8 40.0 1871.3 Harvest 3 46.9 40.0 1874.6 Harvest 4 46.9 40.0 1878.0Harvest 5 47.0 40.0 1881.3 Harvest 6 47.1 40.0 1884.7 BiomassProductivity (g/L/day) 22.5 EPA Content of Biomass (%) 2.3 EPAProductivity (mg/L/h) 21.6 Day 3 Biomass DCW (g/L) Harvest (L) Collected(g) Harvest 1 47.2 40.0 1888.0 Harvest 2 47.6 40.0 1903.0 Harvest 3 48.040.0 1918.2 Harvest 4 48.3 40.0 1933.5 Harvest 5 48.7 40.0 1948.8Harvest 6 49.1 40.0 1964.4 Biomass Productivity (g/L/day) 23.1 EPAContent of Biomass (%) 2.6 EPA Productivity (mg/L/h) 25.0 Day 4 BiomassDCW (g/L) Harvest (L) Collected (g) Harvest 1 49.5 38.0 1881.0 Harvest 250.3 38.0 1909.7 Harvest 3 51.0 38.0 1938.7 Harvest 4 51.8 38.0 1968.3Harvest 5 52.6 38.0 1998.3 Harvest 6 53.4 38.0 2028.7 BiomassProductivity (g/L/day) 24.4 EPA Content of Biomass (%) 2.5 EPAProductivity (mg/L/h) 25.4 Day 5 Biomass DCW (g/L) Harvest (L) Collected(g) Harvest 1 54.2 38.0 2059.6 Harvest 2 53.6 38.0 2037.5 Harvest 3 53.038.0 2015.6 Harvest 4 52.5 38.0 1994.0 Harvest 5 51.9 38.0 1972.5Harvest 6 51.4 38.0 1951.4 Biomass Productivity (g/L/day) 25.1 EPAContent of Biomass (%) 2.2 EPA Productivity (mg/L/h) 23.0 Day 6 BiomassDCW (g/L) Harvest (L) Collected (g) Harvest 1 50.8 38.0 1930.4 Harvest 249.9 38.0 1896.0 Harvest 3 49.0 38.0 1862.1 Harvest 4 48.1 38.0 1828.9Harvest 5 47.3 38.0 1796.3 Harvest 6 46.4 38.0 1764.3 BiomassProductivity (g/L/day) 23.1 EPA Content of Biomass (%) 2.2 EPAProductivity (mg/L/h) 21.2

Example 7—Instructions for Larger Scale Culture

A culture of Nitzschia laevis strain In1CS20 is grown in 70,000 L ofmedia in a 80,000 L stirred tank fermenter. pH is maintained at 8.0 orabove by the addition of KOH, temperature is maintained at 20° C., andpressure at around 300 mbar above surroundings. Agitation is adjusted tokeep the dissolved oxygen levels above 30% saturation at the 60,000 Lpoint in the vessel.

The cells are grown up to a density of around 40 g dry cell weight perliter of culture and harvest initiated. Every 4 hours, around 7000liters of culture are withdrawn from the vessel as harvest usingpressure as a motive force and the volume made back up to 70,000 L withfresh medium. This is sustained for 21 days and produces the equivalentof around 35 tons dry weight of algal biomass or 24 g/L/day over thecourse of the period.

In addition to the media refills, sodium metasilicate solution isprovided to the culture as a separate, continuous, feed such that it isprovided at around 7.5% w/w sodium metasilicate (pentahydrate)/dry cellweight.

Example 8—Ascertaining Nutrient (Sulfur) Levels to Support Log PhaseGrowth of the Diatom

Fully defined growth medium lacking sulfur was made up in which allnutrients apart from sulfur were supplied in non-limiting amounts at pH8.0.

A series of duplicate sterile 250 mL Ehrlenmeyer flasks were prepared byadding 100 mL of the sulfur-free medium to which varying amounts ofmagnesium sulfate had been added. The upper concentration of sulfur waschosen to represent an expected slight excess requirement of the cellswith other concentrations representing 40%, 30%, 20%, 10% and 0% of thislevel.

The flasks were then inoculated with a low level of exponentiallygrowing Nitzschia laevis cells (5 mg dry weight per flask) to limitcarry-over of sulfur from the previous culture. The flasks were placedon an orbital shaker at 20° C. to grow.

Culture dry weights were determined on days 3 to 8 of the culture. Asidefrom the culture with no added sulfur, similar levels of growth wereseen up to day 4 of the culture, at which point cell densities divergedwith those cultures receiving more sulfur growing to higher densities.(See FIG. 2).

By plotting peak dry cell weight against sulfur provision (FIG. 3), itwas determined that the inoculum provided sufficient sulfur to allowbiomass to accumulate to around 1.5 g/L but thereafter approximately amaximum of around 98.5 mg of dry cell weight could be produced per mg ofsulfur. This allowed the determination of the minimum amount of sulfurrequired to support growth of biomass in a traditional batchfermentation (10.1 mg S per g dry cell weight).

However, it is clear from FIG. 2 that the cultures exit log phase growthearlier than they reach maximum dry weight. To calculate the amount ofsulfur required to keep the cells in log phase growth, first the trueamount of sulfur in the culture was calculated using the amount ofbiomass accumulated in the zero sulfur culture and the per dry cellweight sulfur requirements. Then, through plotting the dry cell weightat which the cultures diverged from log phase growth against thecorrected sulfur content of the medium (FIG. 4) it was determined thatonly 91.9 mg of dry cell weight could be kept in log phase growth per mgof sulfur. This translates to 10.9 mg of sulfur being required per g ofdry cell weight to keep the culture in log phase.

In cultures with densities of 45 g DCW/L this difference of around 8%amounts to an additional requirement of around 36 mg/L of sulfur or over600 mg per liter of magnesium sulfate heptahydrate, thus demonstratinghow the difference in approach is critical in medium design.

In order to determine a medium design to support log phase growth, thisexperiment was carried out for each of the nutrients in the medium.Thus, once in possession of the teaching in the present application amedium can be designed following this example, to support log phasegrowth of a variety of diatoms at different scales of fermentation.

Example 9—Instructions for Use of Cyclotella cryptica

The diatom, Cyclotella cryptica is cultured heterotrophically in acontinuous culture in a 14 L fermentor. The medium composition isaccording to Pahl et al. (J Bioscience and Bioengineering 109: 235-239(2010)) adjusted as per the methods of Example 8.)). The pH ismaintained between 7.2 and 8.1 and the temperature between 23 C and 25C. Culture dry cell weight is allowed to increase to at least 40 g dryweight/L at which time a 4 hr cycle of 10% harvest and refill of theculture is initiated. Silicate and fresh medium was fed to the cultureas in Example 1. The EPA content of the biomass is expected to be atleast 2% of dry cell weight. The harvested culture is concentrated bycentrifugation to at least 150 g dry wt/L and freeze dried. The freezedried biomass is extracted with ethanol and the lipids in the ethanolextract are transesterified to form the FAEEs. The FAEEs are partiallyconcentrated by short path distillation (removal of colored compounds)and then the EPA-EE is purified to at least 60% purity by moleculardistillation.

Example 10—Instructions for Use of Phaeodactylum tricornutum

The diatom Phaeodactylum tricornutum (an obligate photoautotroph) isfirst transformed according to the method of Apt et al. (U.S. Pat. No.7,939,710) to give it the ability to grow heterotrophically usingglucose as a source of energy and carbon. The transformed organism iscultured heterotrophically in a continuous culture in a 14 L fermentorusing the culture medium described by Apt (U.S. Pat. No. 7,939,710)adjusted as per the methods of Example 8.). The pH is initially set andmaintained above 8.0. The temperature maintained at 28 C and theagitation and aeration are adjusted to maintain a dissolved oxygen levelof at least 40% of air saturation. The culture dry cell weight isallowed to increase to at least 20 g dry weight/L at which time a 4 hrcycle of 10% harvest and refill of the culture is initiated. Silicateand fresh medium was fed to the culture as in Example 1. The EPA contentof the biomass is expected to be at least 2% of dry cell weight. Theharvested culture is concentrated by centrifugation to at least 150 gdry wt/L and freeze dried. The freeze dried biomass is extracted withethanol and the lipids in the ethanol extract are transesterified toform the FAEEs. The FAEEs are partially concentrated by short pathdistillation and then the EPA-EE is purified to at least 50% purity bymolecular distillation.

Culture dry cell weight and harvest regime are chosen to produce biomassat a rate of at least 20 g dry weight/L/day. Medium conditions arechosen so that the biomass produced contains EPA at a level of greaterthan 2% of dry cell weight.

The invention claimed is:
 1. A method of producing a diatom biomass, themethod comprising the step of continuously culturing a diatom in aculture medium to produce a volumetric production rate of biomass of atleast 20 g dry weight/L/day, wherein the culture medium is designed toprovide the essential nutrients to maintain the diatom in log phasegrowth.
 2. The method of claim 1, wherein the diatom produces at leastone highly unsaturated fatty acid (HUFA).
 3. The method of claim 2,wherein the HUFA is selected from any one or more of EPA, DHA and ARA.4. The method of claim 2, wherein the diatom biomass contains EPA at alevel of at least 2% of dry cell weight of the biomass.
 5. The method ofclaim 1, wherein the mean volumetric production rate of biomass is atleast 20 g dry weight/L/day over a period of at least a month.
 6. Themethod of claim 1, wherein the diatom is selected from any one of aNitzschia species, a Cyclotella species, or a Phaeodactylum species. 7.The method of claim 1, wherein the diatom is cultivatedheterotrophically.
 8. The method of claim 1, wherein the method includesreplacement of at least 50% of the culture volume with fresh mediumevery 24 hr.
 9. A method of producing a diatom biomass, the methodcomprising: a) cultivating a diatom in a culture medium to produce anactive culture; b) removing a portion of the active culture to collectthe biomass; c) adding fresh culture medium to the remaining activeculture that is substantially equivalent in volume to the portion of theactive culture removed in step (b) and further cultivating the diatom;and d) repeating step (b) and step (c) as desired so that at least 20 gdry cell weight of biomass per liter of active culture is collected in a24 hour period, wherein the culture medium is designed to provide theessential nutrients to maintain the diatom in log phase growth.
 10. Themethod of claim 9, wherein the diatom produces at least one highlyunsaturated fatty acid (HUFA).
 11. The method of claim 10, wherein theHUFA is selected from any one or more of EPA, DHA and ARA.
 12. Themethod of claim 11, wherein the diatom microbial biomass contains EPA ata level of at least 2% of dry cell weight of the biomass.
 13. The methodof claim 9, wherein at least 20 g dry cell weight of biomass iscollected per liter of active culture each day for at least sevenconsecutive days.
 14. The method of claim 9, wherein the density ofbiomass in the active culture is at least 20 g/L.
 15. The method ofclaim 9, wherein the diatom is selected from any one of a Nitzschiaspecies, a Cyclotella species, or a Phaeodactylum species.
 16. Themethod of claim 9, wherein the diatom is cultivated heterotrophically.17. The method of claim 9, wherein steps (b) and (c) are carried outsequentially.
 18. The method of claim 9, wherein steps (b) and (c) arecarried out simultaneously.
 19. The method of claim 9, wherein themethod includes the removing of a portion of the active culture in step(b) and the adding of fresh culture medium in step (c) such that thereis replacement of at least 50% of the active culture volume every 24hours.